Abstract 11571: Whole Exome Sequencing in Familial Hypobetalipoproteinemia
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Background: Whole exome sequencing (WES) has shown ~30% success in the diagnosis of Mendelian disorders. Few data exists regarding clinical application of WES for the molecular diagnosis of familial hypobetalipoproteinemia (FHBL), which is characterized as extremely low LDL cholesterol level. Methods: WES was performed on 36 individuals including 32 patients exhibiting low LDL-C (less than 70 mg/dl) primarily, and 4 unaffected family members from 23 families. We filtered out the following variants: 1) Benign variants predicted by SnpEff; 2) Minor allele frequency (MAF) > 1%; 3) Segregation unmatched for the autosomal codominant pattern; 4) C-score < 10 calculated using in silico prediction software named Combined Annotation Dependent Depletion. Results: Among 181,404 variants found in those individuals, we found 48,786 nonsense, missense, or splice site variants, of which 14,415 were rare (MAF ≤ 1% or not reported). Filtering assuming autosomal codominant pattern of inheritance combined with the use of C-score, we identified heterozygous mutations in 7 families, and homozygous or compound heterozygous mutations in 4 families within the coding region of APOB gene, eight of which were novel (c.394A>T/p.Lys132*, c.1902_1903delTC/p.Ser634fs, c.2702T>G/p.Met901Arg, c.2946delC/p.Ser982fs, c.4437G>C/p.Leu1479Phe, c.4439_4440delTT/p.Phe1480fs, c.11283C>A/p.Cys3761*, c.11433dupT/p.Glu3812fs). Moreover, we identified compound heterozygous mutations in 1 family within the coding region of PCSK9 gene, one of which was novel (c.1301G>A/p.Arg434Gln). Conclusion: WES combined with integrated variant annotation prediction successfully identified causative mutations in patients with FHBL either with APOB gene mutation(s) or PCSK9 gene mutation(s) in 12 among 23 families (52%). Although such comprehensive approach is useful to determine true causative mutations, other strategies are needed to identify novel causative genes, which could potentially lead to the development of novel pharmacological target for dyslipidemia.Keywords:
Compound heterozygosity
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Mendelian inheritance
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It is estimated that approximately 85% of human disease mutations are located in protein coding regions, therefore selectively sequencing all protein coding regions (exome) would be cost-effective and an alternative strategy to identify diseases' varaints. In 2009, scientists successfully identified one missense mutation in MYH3 among 4 individuals with Freeman Sheldon syndrome (one autosomal dominant disease) through exome sequencing. Since then, exome sequencing has been widely used to identify disease causative or susceptibility genes in Mendelian disorders and complex diseases. The application of exome sequencing in human diseases were summarized in this review.
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Advances in technology are rapidly changing the field of medical genetics in both the research laboratory and the clinic. With the use of next-generation, or massively parallel, DNA sequencing, it is possible to determine the sequence of essentially all genes in an individual's genome — referred to as the exome — within a matter of days.This technology became widely available in 2005, and the first proof-of-principle experiment showing the power of exome sequencing for the discovery of genes associated with disease was published a few years later.1 Since then, exome analysis has been used in the research setting to . . .
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Abstract Consisting of only ~2% of the human genome, the exome accounts for ~85% of genetic disorders. Efficient sequencing of the human exome with complete and high coverage depth at low cost is invaluable for furthering research in clinical applications. IDT's xGen Exome Panel has proven to be a high performing option. Here, we present the updated xGen Exome Research Panel v2.0 in direct comparison with two other leading commercial human exome panels, using workflows per manufacturer's specifications. NA12878 genomic DNA libraries were pooled together for 8-plex captures for all three platforms and sequenced on the Illumina NextSeq 500. Equivalent number of reads per sample were analyzed against a universal human exome target space to compare across the different exome panels. IDT's Exome NGS solution provided significantly highest on-target percentage at >90% as well as the greatest depth of coverage at >96% bases covered at >20X and >98% bases covered at >10X. Importantly, IDT's platform also reported the most complete gene-level coverage, demonstrated by minimal exon drop-outs in difficult-to-target genes. While 8-plex is the upper limit supported by other suppliers, IDT's platform supports 12-plex workflow. The higher multiplex in combination with high coverage and on-target performance enables IDT to present the lowest total sequencing cost per sample. Since IDT hybridization capture baits are individually synthesized and qualified with the same high standards as standalone oligonucleotide products, lot-to-lot variability is negligible. This presents researchers with an option they can rely on for long-term use and places the focus on the true variabilities of the sample. In conclusion, this study demonstrates xGen Exome Research Panel v2.0, when combined with IDT's DNA Library Prep Kit, provides researchers with a complete Exome NGS solution that is competitive both in performance and sequencing cost. Citation Format: Manqing Hong, Bosun Min, Nicole Roseman, Ekaterina Star, Timothy Rusch, Krishnalekha Datta, Steve Groenewold, Longhui Ren, Jinglie Zhou, Kevin Lai, Xiaohui Wang, Nick Downey, Kristina Giorda, Alexandra Wang, Yu Wang, Lynette A. Lewis, Patrick J. Lau, Steven Henck. Improved human exome sequencing workflow with the most complete coverage [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1349.
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Abstract Purpose: This study was designed to identify the underlying molecular genetic cause of idiopathic hypogonadotropic hypogonadism (IHH) in a nonconsanguineous Chinese family. Methods: All the family members underwent medical history evaluation, physical examination, and laboratory studies. Whole-exome sequencing and RNA sequencing was performed on 2 affected siblings and unaffected parents. All candidate variants were confirmed in all family members by Sanger sequencing and silico function prediction. Results: The proband and his twin brother were diagnosed with IHH, and an autosomal recessive mode of inheritance was identified. Whole-exome sequencing identified the compound heterozygous variants c.9230G>A (p.Arg3077Gln) and c.12883G>A (p.Val4295Met) in DNAH5 in both affected siblings. Sanger sequencing verified that c.9230G>A in DNAH5 was from the unaffected mother and c.12883G>A was from the unaffected father. In addition, we found heterozygous mutations in BBS2 (c.1705delC, p.Gln569fs) and NR5A1 (c.460G>A, p.Ala154Thr) in the siblings, which were from their father. Conclusion: Compound heterozygous variants in DNAH5 were identified in the affected siblings, and were predicted to be pathogenic by silico analysis. This is the first report that suggests variants in DNAH5 are relevant to the pathogenesis of IHH. This study expands the variant spectrum of genes associated with IHH.
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Sequencing the coding regions, the exome, of the human genome is one of the major current strategies to identify low frequency and rare variants associated with human disease traits. So far, the most widely used commercial exome capture reagents have mainly targeted the consensus coding sequence (CCDS) database. We report the design of an extended set of targets for capturing the complete human exome, based on annotation from the GENCODE consortium. The extended set covers an additional 5594 genes and 10.3 Mb compared with the current CCDS-based sets. The additional regions include potential disease genes previously inaccessible to exome resequencing studies, such as 43 genes linked to ion channel activity and 70 genes linked to protein kinase activity. In total, the new GENCODE exome set developed here covers 47.9 Mb and performed well in sequence capture experiments. In the sample set used in this study, we identified over 5000 SNP variants more in the GENCODE exome target (24%) than in the CCDS-based exome sequencing.
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Reporting clinically actionable incidental genetic findings in the course of clinical exome testing is recommended by the American College of Medical Genetics and Genomics (ACMG). However, the performance of clinical exome methods for reporting small subsets of genes has not been previously reported.In this study, 57 exome data sets performed as clinical (n = 12) or research (n = 45) tests were retrospectively analyzed. Exome sequencing data was examined for adequacy in the detection of potentially pathogenic variant locations in the 56 genes described in the ACMG incidental findings recommendation. All exons of the 56 genes were examined for adequacy of sequencing coverage. In addition, nucleotide positions annotated in HGMD (Human Gene Mutation Database) were examined.The 56 ACMG genes have 18 336 nucleotide variants annotated in HGMD. None of the 57 exome data sets possessed a HGMD variant. The clinical exome test had inadequate coverage for >50% of HGMD variant locations in 7 genes. Six exons from 6 different genes had consistent failure across all 3 test methods; these exons had high GC content (76%-84%).The use of clinical exome sequencing for the interpretation and reporting of subsets of genes requires recognition of the substantial possibility of inadequate depth and breadth of sequencing coverage at clinically relevant locations. Inadequate depth of coverage may contribute to false-negative clinical exome results.
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